In this study, SPR based nanosensors were prepared for the detection of amoxicillin (AMOX) in milk samples with using molecular imprinting technique. This study developed to determine the amoxicillin (AMOX) from the commercial and local milk samples. For determination of amoxicillin, polymeric film and nanoparticles consisting of poly(hydroxyethyl methacrylate-N-methacryloyl-(L)- glutamic acid) were synthesized onto the surface of the SPR chip by ultra violet polymerization. The characterization of the polymeric film and nanoparticles were determined by zeta sizer, ellipsometry, contact angle and atomic force microscopy measurements. The range of linearity was measured as 0.1-200 ng/mL for amoxicillin (AMOX) imprinted polymeric film and nanoparticles based SPR nanosensors. The limit of detection for AMOX imprinted polymeric film and nanoparticles based SPR nanosensors were found as 0.0012 ng/mL and 0.0009 ng/mL, respectively. To show the selectivity of surface plasmon resonance nanosensors competitive adsorption of amoxicillin (AMOX), ampicillin (AMP), cephalexin (CEP) and cholesterol (KOL) were investigated. According to the results, AMOX imprinted SPR nanosensors have exhibited low-detection limit, high sensitivity and selectivity for detection of antibiotic in milk samples.